Diastolic dysfunction can impair ventricular filling, and can be a major contributor to the cardiac dysfunction in heart failure. In the clinic, approximately half of patients with congestive heart failure (CHF) present mainly with diastolic dysfunction. It stands to reason that in order to understand the mechanism governing diastolic dysfunction, it would first be necessary to understand how myocardial relaxation takes place in the non-pathological setting. We hypothesize that myofilament properties, rather than calcium transient decline, primarily determines duration of relaxation in adult mammalian myocardium. To test this hypothesis, we simultaneously measured force of contraction and calibrated [Ca2+]i transients in isolated, thin rabbit right ventricular cardiac trabeculae (n = 8), at various lengths at 37 ºC. Time from peak tension to 50% relaxation (RT50(tension)) increases significantly with length (from 49.8 ± 3.4 ms to 83.8 ± 7.4 ms at an [Ca2+]o of 2.5 mM), while time from peak calcium to 50% decline (RT50(calcium)) was not prolonged (from 124.8 ± 5.3 ms to 107.7 ± 11.4 ms at an [Ca2+]o of 2.5 mM). Analysis by two-way ANOVA revealed that RT50(tension) is significantly correlated with length (P < 0.0001). At optimal length, varying the extracellular calcium concentration increased both developed force and calcium transient amplitude, but RT50(tension) remained unchanged (P = 0.90), while intracellular calcium decline actually accelerated (P < 0.05). Thus, an increase in muscle length will result in an increase in both force and duration of relaxation, while the latter is not primarily governed by the rate of [Ca2+]i decline. Our data provide further insight into the mechanism of myocardial relaxation, and the results show that increasing muscle length significantly dissociates intracellular calcium transient decline from force decline. In conclusion, when investigating cardiac relaxation disorders, myofilament properties need to be prominently considered for strategizing future treatment options.